A synthetic HIV-1 protease inhibitor with antiviral activity arrests HIV-like particle maturation.

A synthetic peptidemimetic substrate of the human immunodeficiency virus 1 (HIV-1) protease with a nonhydrolyzable pseudodipeptidyl insert at the protease cleavage site was prepared. The peptide U-81749 inhibited recombinant HIV-1 protease in vitro (inhibition constant Ki of 70 nanomolar) and HIV-1 replication in human peripheral blood lymphocytes (inhibitory concentration IC50 of 0.1 to 1 micromolar). Moreover, 10 micromolar concentrations of U-81749 significantly inhibited proteolysis of the HIV-1 gag polyprotein (p55) to the mature viral structural proteins p24 and p17 in cells infected with a recombinant vaccinia virus expressing the HIV-1 gag-pol genes. The HIV-1 like particles released from inhibitor-treated cells contained almost exclusively p55 and other gag precursors, but not p24. Incubation of HIV-like particles recovered from drug-treated cultures in drug-free medium indicated that inhibition of p55 proteolysis was at least partially reversible, suggesting that U-81749 was present within the particles.

The location of v-src in a retrovirus vector determines whether the virus is toxic or transforming.

We prepared infectious stocks of an avian retrovirus, a modified spleen necrosis virus, containing the herpes simplex virus type 1 thymidine kinase gene and the avian sarcoma virus v-src gene. Viruses were recovered after cotransfection of chicken cells with DNA of recombinants between cloned spleen necrosis virus thymidine kinase and v-src and with DNA of cloned reticuloendotheliosis virus strain A. When v-src was inserted near the 5'end of the viral genome, only low titers of recombinant virus were recovered. Most of the recovered viruses were smaller than expected and did not transform the morphology of rat or chicken cells. A very small amount of virus of the expected structure was recovered; this virus transformed rat cells and expressed v-src. Cotransfection data indicated that one reason we failed to recover a significant titer of recombinant virus is that efficient expression of v-src is acutely toxic to chicken and dog cells. Insertion of v-src near the 3' end of the viral genome, such that it was expressed at a lower level compared with the 5'-v-src-containing virus, yielded a higher titer of recombinant virus, and this virus was transforming. The differences in the recovery and transforming activity of these viruses indicate that the location of an oncogene in the viral genome is an important factor regulating the level of its expression and whether or not this expression is toxic or transforming to cells.

Adducts from the reaction of N-benzoyloxy-N-methyl-4-aminoazobenzene with deoxyguanosine or DNA in vitro and from hepatic DNA of mice treated with N-methyl- or N,N-dimethyl-4-aminoazobenzene.

N-Benzoyloxy-N-methyl-4-aminoazobenzene, a synthetic model ultimate carcinogenic derivative of N-methyl- and N,N-dimethyl-4-aminoazobenzene, reacted with [8-14C]deoxyguanosine in vitro to yield ten 14C-containing products separable by high-performance liquid chromatography. N-(Deoxyguanosin-8-yl)-N-methyl-4-aminoazobenzene, previously characterized as the major adduct formed in this reaction, occurred as cis and trans isomers which were interconvertible and together accounted for 75% of the reaction of N-benzoyloxy-N-methyl-4-aminoazobenzene with [8-14C]deoxyadenosine. The products obtained by reaction of DNA with N-benzoyloxy-N-methyl-4-aminoazobenzene and subsequent digestion to deoxynucleosides had a profile similar to that of the products obtained from deoxyguanosine except that the DNA digest contained a second major product that was formed in only small amounts on reaction with deoxyguanosine. The latter product has been identified as 3-(deoxy-guanosin-N2-yl)-N-methyl-4--aminoazobenzene by Beland et. al. (Chem.-Biol. Interactions, in press). Reaction with [purine-14C]DNA showed that most, if not all, of the products were derived from purine nucleosides. Digests of the DNA from the livers of C57BL/6 X C3H/He F1 mice, given injections at 12 days of age with [prime ring-3H]N-methyl- or N,N-dimethyl-4-aminoazobenzene, contained the same major adducts as did those obtained from DNA reacted with N-benzoyloxy-N-methyl-4-aminoazobenzene. The hepatic DNA also yielded a series of minor products that comigrated on high-performance liquid chromatography with adducts obtained in the reactions with N-benzoyloxy-N-methyl-4-aminoazobenzene. Approximately 70 and 30%, respectively, of the N-(deoxyguanosin-8-yl)-N-methyl-4-aminoazobenzene and of the second major product were removed from the mouse liver DNA within 10 days after administration of [prime ring-3H]N,N-dimethyl-4-aminoazobenzene. These data are of interest in view of the development of multiple hepatic tumors in mice of this hybrid treated prior to weaning with N-methyl- or N,N-dimethyl-4-aminoazobenzene.

4-aminoazobenzene and N,N-dimethyl-4-aminoazobenzene as equipotent hepatic carcinogens in male C57BL/6 X C3H/He F1 mice and characterization of N-(Deoxyguanosin-8-yl)-4-aminoazobenzene as the major persistent hepatic DNA-bound dye in these mice.

In contrast to the well-established requirement for an N-methyl group for efficient hepatic tumor induction by dietary administration of derivatives of 4-aminoazobenzene (AB) to adult rats, we have now observed that AB and its N-methyl and N,N-dimethyl derivatives have high and approximately equal hepatocarcinogenicity when given as a single i.p. dose to male 12-day-old C57BL/6 X C3H/ HeF1 (B6C3F1) mice. The hepatoma multiplicity induced by these dyes was approximately linearly related to the dose from 0.017 to 0.15 mumol/g body weight; at the high dose, an average of 11 hepatomas/mouse was observed at 10 months. Female B6C3F1 mice were resistant to tumor induction under these conditions. AB and its N-methyl derivative also induced the same incidences of hepatomas on administration of a single dose of 0.45 mumol/g body weight to 12-day-old male C3H/He mice (about 15 hepatomas/mouse) or C57BL/6 mice (about 1 hepatoma/mouse). Infant male Fischer rats were much less susceptible; less than 25% of the rats given 4 i.p. injections (0.3 to 0.4 mumol/g of body weight/injection) of N-methyl-4-amino-azobenzene and less than or equal to 5% of those given these doses of N,N-dimethyl-4-aminoazobenzene or AB before 22 days of age developed hepatic carcinomas by 24 months. Reverse-phase high-performance liquid chromatography of enzymatically hydrolyzed hepatic DNA from 12-day-old male B6C3F1 mice or Fischer rats given an i.p. dose (0.08 or 0.3 mumol/g of body weight) of [prime-ring-3H]AB showed a single major adduct which was chromatographically identical to N-( deoxyguanosin -8-yl)-4-aminoazobenzene synthesized by reaction at pH 7 of N-acetoxy-4-aminoazobenzene (formed in situ from N-hydroxy-4-aminoazobenzene and acetic anhydride) with deoxyguanosine. Mouse and rat liver DNA contained 20 and 0.5 pmol, respectively, of this adduct per mg 24 hr after administration of 0.3 mumol of [prime-ring-3H]AB/g of body weight. At 24 hr after administration of N,N-[prime-ring-3H]dimethyl-4-aminoazobenzene to male B6C3F1 mice, N-( deoxyguanosin -8-yl)-4-aminoazobenzene, N-( deoxyguanosin -8-yl)-N-methyl-4-aminoazobenzene, and 3-( deoxyguanosin -N2-yl)-N-methyl-4-aminoazobenzene were present in a ratio of approximately 4:2:1, respectively. Unlike the N-( deoxyguanosin -8-yl)-N-methyl-4-aminoazobenzene adducts, the N-( deoxyguanosin -8-yl)-4-aminoazobenzene adducts were relatively stable in the DNA; the level of the latter adducts decreased about 60% between 24 hr and 21 days.(ABSTRACT TRUNCATED AT 400 WORDS)

Oxazolidinones: a new class of antibacterials.

The oxazolidinones represent the first truly new class of antibacterial agents to reach the marketplace in several decades. They have a unique mechanism of action involving inhibition of the initiation step of protein synthesis and are not cross-resistant to other classes of antibiotics. The first marketed member of that class, linezolid (Zyvox), shows good efficacy with an impressive antibacterial spectrum (including activity against gram-positive organisms resistant to other drugs), and a pharmacodynamic/pharmacokinetic relationship best characterized by time above the minimum inhibitory concentration. The agent is effective by both the intravenous and oral route of administration. Although technically classified as bacteriostatic against a number of pathogens in vitro, linezolid behaves in vivo like a bactericidal antibiotic.

Secretion of enzymatically active human renin from mammalian cells using an avian retroviral vector.

Recombinant plasmid-based retroviral expression vectors were constructed using a modified spleen necrosis virus (SNV) containing the Herpes simplex virus thymidine kinase gene promoter controlling the expression of the Tn5 neomycin phosphotransferase II gene (NPTII gene). The human renin (HRn) gene (hrn) was inserted into the 5' end of the SNV sequences such that in concatemeric plasmid DNA its expression was controlled by the strong promoter in the SNV long terminal repeat (LTR). Dog cells transfected with the concatemeric plasmid DNA secreted a small amount of a HRn-like 43-kDa protein. After cotransfection of chicken cells with concatemeric plasmid DNA and proviral DNA of reticuloendotheliosis virus strain A, infectious stocks of viruses were recovered. Cells infected with the virus carrying the viral LTR-hrn gene oriented for expression secreted the 43-kDa HRn-like protein at about 100-fold higher levels than the cells transfected with the plasmid DNAs. Biological activity of secreted HRn was determined by measuring levels of angiotensin I generated by incubating culture media with either a porcine or human angiotensinogen substrate. Infected dog cells produce about 40 ng of enzymatically active HRn per 10(6) cells per 24 h. These data indicate that retroviral expression vectors provide a good system for obtaining the secretion of high levels of enzymatically active heterologous proteins from mammalian cells.

Mechanism of resistance to U-90152S and sensitization to L-697,661 by a proline to leucine change at residue 236 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase.

Bisheteroarylpiperazines (BHAPs) are highly specific inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). BHAP-resistant HIV-1 is sensitized to other classes of nonnucleoside RT inhibitors and this has been primarily attributed to a proline-to-leucine substitution at amino acid 236 (P236L) of HIV-1 RT. To understand the basis for the in vitro sensitization-resistance phenomenon, single base pair mutations at amino acid P236 in HIV-1 RT were introduced to obtain P236L, P236T, P236H, P236R, and P236A HIV-1 RT mutants. Active HIV-1 RT mutants H235W, D237T, and H235W/D237T/T240K, containing substitutions from HIV-2 RT, were also cloned, expressed, and purified. Three BHAPs (U-88204E, U-87201E, and U-90125S) and the pyridinone L-697,661 were selected to quantitatively assess the effects of these amino acid substitutions on sensitization to L-697,661 and resistance to the BHAPs. The HIV-1 RT mutants bearing single (H235W; D237T) or multiple (H235W/D237T/T240K) HIV-2 RT substitutions around the conserved P236 conferred little resistance or sensitization to these RT inhibitors. The inhibition profiles of the P236 HIV-1 RT mutants demonstrated a direct correlation between sensitization to L-697,661 and resistance to the BHAPs. These results suggest alterations in the shape of the binding pocket as the mechanism by which the P236L mutation confers resistance to the BHAPs and sensitization to L-697,661.

Simultaneous mutations at Tyr-181 and Tyr-188 in HIV-1 reverse transcriptase prevents inhibition of RNA-dependent DNA polymerase activity by the bisheteroarylpiperazine (BHAP) U-90152s.

The replacement of either Tyr-181 or Tyr-188 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) by the corresponding HIV-2 RT amino acids Ile-181 or Leu-188 is known to result in active mutant enzymes (Y181I; Y188L) with virtual loss of sensitivity towards three structural classes of nonnucleoside RT inhibitors; L-697,661, nevirapine, and TIBO R82913. The bisheteroarylpiperazine (BHAP) U-90152S, a highly specific inhibitor (IC50, 0.29 +/- 0.01 microM) of HIV-1 RT, inhibited the recombinant Y181I and Y188L HIV-1 RT mutants with IC50 values of 3.6 +/- 0.15 microM and 0.71 +/- 0.02 microM, respectively. Construction and in vitro analysis of double mutants Y181I/Y188L and Y181C/Y188L of HIV-1 RT showed > 150-fold resistance to U-90152S. An HIV-2 RT mutant containing amino acids 176-190 from HIV-1 RT acquired full sensitivity to U-90152S (IC50, 0.26 +/- 0.01 microM). It is concluded that simultaneous mutations at Tyr-181 and Tyr-188 of HIV-1 RT promotes resistance to U-90152S.

Metabolism of cis-delta4-15(S)-15-methylprostaglandin F1alpha methyl ester in the rat.

The metabolic transformation and excretion of tritium-labeled cis-delta4-15(S)-15-methylprostaglandin F1alpha methyl ester (1) have been investigated in rats after chronic and single dose oral administration. Three metabolites have been identified from a partially purified urinary extract. They were the cis-delta4-15-methylprostaglandin F1alpha (2), 15-methyl-2,3-dinorprostaglandin F1alpha (3), and 15-methyl-2,3,4,5-tetranorprostaglandin F1alpha (4). The excretion of the drug-related species was significantly slower than that of the natural prostaglandin F2alpha. The slow excretion rate and the presence of unchanged cis-delta4-15-methylprostaglandin F1alpha in urine suggested that the metabolically protected prostaglandin could persist in the body and, therefore, exhibit longer duration of action.

Novel 1,5-diphenylpyrazole nonnucleoside HIV-1 reverse transcriptase inhibitors with enhanced activity versus the delavirdine-resistant P236L mutant: lead identification and SAR of 3- and 4-substituted derivatives.

Through computationally directed broad screening, a novel 1, 5-diphenylpyrazole (DPP) class of HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs) has been discovered. Compound 2 (PNU-32945) was found to have good activity versus wild-type (IC(50) = 2.3 microM) and delavirdine-resistant P236L (IC(50) = 1.1 microM) reverse transcriptase (RT). Also, PNU-32945 has an ED(50) for inhibition of viral replication in cell cultures of 0.1 microM and was shown to be noncytotoxic with a CC(50) > 10 microM. Structure-activity relationship studies on the 3- and 4-positions of PNU-32945 led to interesting selectivity and activity within the class. In particular, the 3-hydroxyethyl-4-ethyl congener 29 is a potent inhibitor of the P236L mutant (IC(50) = 0.65 microM), whereas it is essentially inactive versus the wild-type enzyme (IC(50) > 50 microM). Furthermore, this compound was significantly more active versus the P236L mutant than delavirdine. The synthesis and RT inhibitory activity of various 3- and 4-substituted analogues are discussed.

Inhibitors of the protease from human immunodeficiency virus: design and modeling of a compound containing a dihydroxyethylene isostere insert with high binding affinity and effective antiviral activity.

The peptidomimetic template and the dihydroxyethylene isostere insert that were applied successfully to the design of renin inhibitors have been extended to the related protease from human immunodeficiency virus (HIV). The present report describes the structure-activity study leading to the identification of an inhibitor with a Ki of less than 1 nM for the HIV type-1 protease (compound II). This compound, containing a diol insert, is highly effective in blocking polyprotein processing in in vitro cell culture assays. Results obtained from kinetic analysis, studies of the stereochemistry of the insert, and modeling have led to insights as to the requisites involved in the active site-inhibitor interaction.

Inhibitors of the protease from human immunodeficiency virus: synthesis, enzyme inhibition, and antiviral activity of a series of compounds containing the dihydroxyethylene transition-state isostere.

A number of potential HIV protease inhibitory peptides that contain the dihydroxyethylene isostere were prepared and evaluated for their enzyme binding affinity and antiviral activity in cell cultures. From the template of a previously reported active peptide A, modifications at the N- and C-terminal groups were assessed for potential maintenance of good inhibitory activity of the resulting peptides. Among the active peptides found, peptide XVIII exhibited potent enzyme inhibitory activity. Interestingly, the previously reported, effective 1(S)-amino-2(R)-hydroxyindan C-terminal group for the preparation of very active HIV protease inhibitory peptides could not be applied to the template of peptide XVIII. Molecular modeling of peptide XVIII was studied using the X-ray crystal structure of peptide A as a starting point in order to study the likely conformation of peptide XVIII in the active-site cleft. Relative binding conformations of peptide A and XVIII were obtained, although the reason for poor binding affinity for a number of congeneric peptides in this report was not straightforwardly apparent. More importantly, however, peptide XVIII was found to exhibit more effective antiviral activity in the HIV-1/PBMC assay than the reference peptide A which was previously reported to be approximately equal in efficacy to the reverse transcriptase inhibitor AZT in this assay.

Pyrimidine thioethers: a novel class of HIV-1 reverse transcriptase inhibitors with activity against BHAP-resistant HIV.

A series of pyrimidine thioethers was synthesized and evaluated for inhibitory properties against wild-type HIV-1 reverse transcriptase (RT) and an RT carrying the resistance-conferring mutation P236L. Modifications of both the pyrimidine and the functionality attached through the thioether yielded several analogues, which demonstrated activity against both enzyme types, with IC50 values as low as 190 nM against wild-type and 66 nM against P236L RT. Evaluation of a select number of pyrimidine thioethers in cell culture showed that these compounds have excellent activity against HIV-1IIIB-WT and retain good activity against a laboratory-derived HIV-1MF delavirdine-resistant variant.

Targeting delavirdine/atevirdine resistant HIV-1: identification of (alkylamino)piperidine-containing bis(heteroaryl)piperazines as broad spectrum HIV-1 reverse transcriptase inhibitors.

A novel class of bis(heteroaryl)piperazine (BHAP) analogs which possesses the ability to inhibit NNRTI (non-nucleoside reverse transcriptase inhibitor) resistant recombinant HIV-1 reverse transcriptase (RT) and NNRTI resistant variants of HIV-1 has been identified via targeted screening. Further investigation of the structure-activity relationships of close congeners of these novel (alkylamino)piperidine BHAPs (AAP-BHAPs) led to the synthesis of several compounds possessing the desired phenotype (e.g., activity against recombinant RTs carrying the Y181C and P236L substitutions). Further structural modifications were required to inhibit metabolism and modulate solubility in order to obtain compounds with the desired biological profile as well as appropriate pharmaceutical properties. The AAP-BHAPs with the most suitable characteristics were compounds 7, 15, and 36.

Escherichia coli expression, purification, and biological activity of a truncated soluble CD4.

A truncated molecule containing the N-terminal 183 amino acid residues of CD4 (sCD4-183) has been produced in Escherichia coli at high levels, using the trp promoter and an AT-rich ribosome binding site to direct expression in a pBR322-derived vector. A culture has been selected which allows large-scale fermentation and production of this material as an insoluble inclusion body protein. Procedures which solubilize, refold, and purify sCD4-183 have been developed. The purified sCD4-183 binds gp120 in solution and blocks human immunodeficiency virus (HIV) infection of human peripheral blood lymphocytes in vitro.

Denaturation/refolding of purified recombinant HIV reverse transcriptase yields monomeric enzyme with high enzymatic activity.

We engineered a prokaryotic expression vector encoding the HIV reverse transcriptase (RT). We grew Escherichia coli JM109 carrying the vector in a 250-liter stirred tank fermentor and purified RT (p66) under native conditions to apparent homogeneity. Purified p66 (greater than or equal to 5 mg/ml) was not stable, and was rapidly processed to its 51 kD derivative (p51), until p66:p51 levels were approximately 1:1. These latter RT preparations were chromatographed as heterodimers and had approximately fivefold higher specific RT enzymatic activities compared with those containing predominantly p66. P66 purified under dilute concentrations (less than or equal to 0.5 mg/ml) was monomeric in solution, resistant to p51 processing for weeks at 4 degrees C, but also had low specific RT enzymatic activities. To attempt the preparation of homogeneous p66 with specific RT enzymatic activities equivalent to p66:p51 heterodimers, purified heterodimers were denatured and p66 was purified and refolded during extensive dialysis (refolded p66). Refolded p66 (less than or equal to 0.5 mg/ml) was monomeric in solution and had identical specific RT enzymatic activities, Km for dTTP, and inhibition by 3'-azido-3'-deoxythymidine triphosphate compared with heterodimeric p66:p51 RT. The data indicates that HIV RT obtained from recombinant E. coli under native conditions is extensively processed at concentrations promoting dimerization. Moreover, RT denaturation and refolding yields apparently homogeneous monomeric p66, with specific RT enzymatic activities equivalent to heterodimeric RT.

A rapid, quantitative bioassay based on the human immunodeficiency virus trans-activator.

We constructed a human immunodeficiency virus (HIV) trans-activator cDNA (tat) encoding the N-terminal 76 amino acids of the viral trans-activator followed by two additional amino acids (val and pro). This cDNA encoded a functional trans-activator (TAT) as shown by cotransfection into murine cells with a HIV promoter-chloramphenicol acetyltransferase DNA construct. The tat cDNA was cloned into an avian retroviral expression vector, a modified spleen necrosis virus (SNV), and high-titer infectious stocks of recombinant virus (SNV-tat) were recovered from dog cells. Hybridization analyses indicated that SNV-tat was stably propagated in these cells for months. We also prepared recombinant cells that stably carry reporter genes, either a human gene encoding a soluble CD4 receptor (sCD4) or the human preprorenin gene, under the transcriptional control of the HIV promoter. Medium obtained from these cell cultures after infection with control viruses or an SNV carrying an antisense tat contained only low background levels of sCD4 or prorenin (HRN) as determined by specific immunoassays (1-10 ng protein per 10(6) cells per ml medium). In contrast, cells infected with SNV carrying tat in the transcriptional sense orientation secreted 75 +/- 7 ng sCD4 and 73 +/- 4 ng HRN per 10(6) cells per ml medium. Moreover, these proteins were constitutively secreted at these levels during months of subculturing. The data indicate that sCD4 and HRN are secreted from these cells because of a TAT-mediated trans-activation of the HIV reporter gene DNA and/or RNA. This combination of recombinant cells, SNV-tat, and specific immunoassays provide a rapid, quantitative, and safe bioassay to seek inhibitors of TAT.

Kinetic studies with the non-nucleoside human immunodeficiency virus type-1 reverse transcriptase inhibitor U-90152E.

The bisheteroarylpiperazine U-90152E is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and possesses excellent anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. The compound inhibits both the RNA- and DNA-directed DNA polymerase functions of HIV-1 RT. Kinetic studies were carried out to elucidate the mechanism of RT inhibition by U-90152E. Michaelis-Menten kinetics, which are based on the establishment of a rapid equilibrium between the enzyme and its substrates, proved inadequate for the analysis of the experimental data. The data were thus analyzed using Briggs-Haldane kinetics, assuming that the reaction is ordered in that the template:primer binds to the enzyme first, followed by the addition of dNTP and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived, which allows the calculation of all the essential forward and backward rate constants for the reactions occurring between the enzyme, its substrates and the inhibitor. The results obtained indicate that U-90152E acts exclusively as a mixed inhibitor with respect to the template: primer and dNTP binding sites for both the RNA- and DNA-directed DNA polymerase domains of the enzyme. The inhibitor shows a significantly higher binding affinity for the enzyme-substrate complexes than for the free enzyme and consequently does not directly impair the functions of the substrate binding sites. Therefore, U-90152E appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either inhibition of the phosphoester bond formation or translocation of the enzyme relative to its template:primer following the formation of the ester bond.

The benzylthio-pyrimidine U-31,355, a potent inhibitor of HIV-1 reverse transcriptase.

U-31,355, or 4-amino-2-(benzylthio)-6-chloropyrimidine is an inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and possesses anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. The compound acts as a specific inhibitor of the RNA-directed DNA polymerase function of HIV-1RT and does not impair the functions of the DNA-catalyzed DNA polymerase or the Rnase H of the enzyme. Kinetic studies were carried out to elucidate the mechanism of RT inhibition by U-31,355. The data were analyzed using Briggs-Haldane kinetics, assuming that the reaction is ordered in that the template:primer binds to the enzyme first, followed by the addition of dNTP, and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived that allows the calculation of all the essential forward and backward rate constants for the reactions occurring between the enzyme, its substrates, and the inhibitor. The results obtained indicate that U-31,355 acts as a mixed inhibitor with respect to the template:primer and dNTP binding sites associated with the RNA-directed DNA polymerase domain of the enzyme. The inhibitor possessed a significantly higher binding affinity for the enzyme-substrate complexes, than for the free enzyme and consequently did not directly affect the functions of the substrate binding sites. Therefore, U-31,355 appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either inhibition of the phosphoester bond formation or translocation of the enzyme relative to its template:primer following the formation of the ester bond. Moreover, the potency of U-31,355 depends on the base composition of the template:primer in that the inhibitor showed a much higher binding affinity for the enzyme-poly (rC):(dG)10 complexes than for the poly (rA):(dT)10 complexes.

Effects of a soluble CD4 and CD4-Pseudomonas exotoxin A chimeric protein on human peripheral blood lymphocytes: lymphocyte activation and anti-HIV activity in vitro.

Recombinant sCD4-based proteins were evaluated for their effects on antigen-stimulated proliferation of human peripheral blood mononuclear cells (PBMC) and for antiviral activity against PBMC infected with human immunodeficiency virus (HIVD34). Two sCD4-based proteins were solubilized, refolded, and purified to homogeneity from recombinant E. coli and consisted of the 178 amino-terminal residues of CD4 fused with the translocating and catalytic domains of Pseudomonas exotoxin A (sCD4-PE40) or 183 amino-terminal residues of CD4 (sCD4-183); a third sCD4 consisting of 369 amino acids of CD4 was purified from recombinant mammalian cells for comparative purposes (sCD4-369). Increasing molar concentrations of these sCD4s were evaluated for inhibition of PBMC proliferation induced by alloantigen (MLR), by tetanus toxoid (TTOX), or in response to crosslinking with antibody to CD3 (OKT3). In addition, the concentrations of each protein required to inhibit replication of the HIVD34 isolate in primary PBMC was determined by quantitation of HIV p24 antigen released into supernatant fluids by infected cells. By comparing antiviral activity with anti-proliferative activity a relative estimate of the selectivity index for each recombinant sCD4 was determined. Proliferation of PBMC in response to alloantigen or OKT3 was less sensitive to inhibition than proliferation induced by TTOX, and the selectivity indices estimated for sCD4-PE40 were 170, 170 and 17, respectively. The selectivity index for sCD4-183 was greater than 350 under all assay conditions. Comparative evaluation of alloantigen-stimulated proliferation with antiviral activity of sCD4-183 versus sCD4-369 suggested that the E. coli-derived sCD4-183 may have a higher selectivity index under these conditions than its mammalian cell-derived counterpart.